About 500,000 Americans die of cancer every year. Much progress has been made in our understanding of cancer biology through the use of animal models. Developing the zebrafish system as a cancer model would create a new, powerful research tool. Zebrafish are vertebrates with a well established history in forward genetics and cancer toxicology and are known to develop neoplasms that resemble human cancers. Members of the Zon lab have developed a strategy to screen mutagenized zebrafish for defects in the embryonic cell cycle, hypothesizing that some of these mutants would be more susceptible to cancer. Seven zebrafish cell cycle mutants have been identified and are embryonic lethal. Adult heterozygotes for five of the seven mutations exhibit an increased cancer prevalence. To further characterize the cell cycle defects, we will utilize chemicals (e.g., nocodazole) to synchronize the cell cycle in wild-type and mutant embryos. Progression of the synchronized cohort through the cell cycle will be observed using cell cycle markers. In addition, we will test chemicals that override checkpoints (e.g., caffeine) for the ability to alter the phenotype of mutants exhibiting cell cycle arrest. Based on this cell cycle characterization and data on cancer susceptibility, one cell cycle/cancer-prone mutant will be chosen for candidate gene or positional cloning. Gene identification would lay the foundation for traditional genetic and chemical genetic strategies for identifying genes or compounds that interact with the pathway. In that regard, a second aim is to use a cell cycle/cancer prone mutant to develop zebrafish as a chemical genetic model system for identifying drugs that perturb oncogenic pathways. This goal will be accomplished in collaboration with two experts in chemical genetics, Timothy Mitchison and Randall King of the Institute of Chemistry and Cell Biology at Harvard Medical School. Zebrafish are ideal for whole-embryo-based small molecule screens because they are water-dwellers that are sensitive to chemicals and give rise to large numbers of small embryos. Intact-embryo-based screens have the advantages of selecting for compounds that are not toxic to multicellular organisms and of avoiding the use of transformed cell lines. Using a whole-organism-based approach, a single screen will be able to detect chemicals that suppress the cell cycle mutant phenotype, that are synthetic lethal with the mutation, or that are selectively toxic. Such molecules will then be examined for the ability to alter tumor rate or size in adult fish. Chemicals that perturb specific oncogenic pathways will be valuable tools for studying tumor progression in the zebrafish cancer model system and could be lead compounds for human cancer chemotherapy and/or chemoprevention.